1. Field of the Invention
This invention relates to a novel bioactive substance which suppresses differentiation of undifferentiated cells.
2. Description of Related Art
Human blood and lymph contain various types of cells and each cell plays important roles. For example, erythrocytes carry oxygen; platelets have hemostatic action; and lymphocytes prevent infection. These various cells originate from hematopoietic stem cells in the bone marrow. Recently, it has been clarified that the hematopoietic stem cells are differentiated to various blood cells, osteoclasts and mast cells by stimulation of various cytokines in vivo and environmental factors. In the cytokines, there have been found, for example, erythropoietin (EPO) for differentiation to erythrocytes; granulocyte colony stimulating factor(G-CSF) for differentiation to leukocytes; and platelet growth factor (mpl ligand) for differentiation to megakaryocytes which are platelet producing cells; and the former two examples have already been clinically applied.
The differentiated blood cells are generally classified into two groups consisting of blood precursor cells which are destined to differentiate to specific blood series and hematopoietic stem cells which have differentiation ability to all series and self-replication activity. The blood precursor cells can be identified by various colony assays; however, an identification method for the hematopoietic stem cells have not been established. In these cells, stem cell factor (SCF), interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-1 (IL-1), granulocyte colony stimulating factor (G-CSF) and oncostatin M have been reported to stimulate cell differentiation and proliferation.
Trials for expansion of hematopoietic stem cells in vitro have been conducted in order to replace bone marrow transplantation for applying hematopoietic stem cell transplantation therapy or gene therapy. However, when the hematopoietic stem cells are cultured in the presence of the above mentioned cytokines, multi-differentiation activities and self-replication activities, which are originally in the position of the hematopoietic stem cells, gradually disappeared and are changed to the blood cell precursors which only differentiate to specific series after 5 weeks of cultivation, and multi-differentiation activity, which is one of the specific features of the hematopoietic stem cells, is lost (Wanger et al. Blood 86, 512-523, 1995).
For proliferation of the blood precursor cells, a single cytokine is not sufficient, but rather the synergistic action of several cytokines is important. Consequently, in order to proliferate the hematopoietic stem cells while maintaining the specific features of the hematopoietic stem cells, it is necessary to add cytokines which suppress differentiation together with the cytokines which proliferate and differentiate the undifferentiated blood cells. In general, many cytokines which stimulate proliferation or differentiation of cells are known, but few cytokines which suppress cell differentiation are known. For example, leukemia inhibitory factor (LIF) has an action of proliferation of mouse embryonic stem cells without differentiation, but it has no action against the hematopoietic stem cells or blood precursor cells. Transforming growthfactor (TGF-.beta.) has suppressive action for proliferation against various cells, but has no fixed actions against the hematopoietic stem cells or blood precursor cells.
Not only blood cells but also undifferentiated cells, especially stem cells, are thought to be involved in tissue regeneration. These regeneration of tissues and proliferation of undifferentiated cells in each tissue can be applied in various known ways (Katsutoshi Yoshizato, Regenration--a mechanism of regeneration, 1966, Yodosha Publ. Co.).
Notch is a receptor type membrane protein involved in regulation of nerve cell differentiation found in Drosophia. Homologues of Notch are found in various invertebrates and vertebrates including nematoda (Lin-12), Xenopus laevis (Xotch), mouse (Motch) and human (TAN-1).
Ligands of Notch in Drosophila are known. These are Drosophila Delta (Delta) and Drosophila Serrate (Serrate). Notch ligand homologues are found in various animals similar to those of Notch receptors (Artavanis-Tsakonas et al., Science 268, 225-232, 1995).
Human Notch homologue, TAN-1 is found widely in the tissues in vivo (Ellisen et al., Cell 66, 649-661, 1991). Two Notch analogous molecules other than TAN-1 have been reported (Artavanis-Tsakonas et al., Science 268, 225-232, 1995). Expression of TAN-1 was also observed in CD34 positive cells in blood cells by PCR (Polymerase Chain Reaction) (Milner et al., Blood 83, 2057-2062, 1994). However, in relation to humans, gene cloning of human Delta and human Serrate, which are thought to be Notch ligand, has not been reported.
In Drosophila Notch, binding with the ligand was studied and investigated in detail, and it was found that the Notch can be bound to the ligand with Ca++ at the binding region, which is a repeated amino acid sequence No. 11 and No. 12 in the amino acid sequence repeat of Epidermal Growth Factor (EGF) (Fehon et al., Cell 61, 523-534, 1990, Rebay et al., ibid. 67, 687-699, 1991 and Japan. Patent PCT Unexam. Publ. 7-4 503123). EGF-like repeated sequences are conserved in Notch homologues of other species. Consequently, the same mechanism in binding with ligand is assumed.
An amino acid sequence which is called DSL (Delta-Serrate-Lag-2) near the amino acid terminal, and EGF-like repeated sequence like in the receptor are conserved in the ligand (Artavanis-Tsakonas et al., Science 268, 225-232, 1995). EGF-like sequence has been found in thrombo-modulin (Jackman et al., Proc. Natl. Acad. Sci. USA 83, 8834-8838, 1986), low density lipoprotein (LDL) receptor (Russell et al., Cell 37, 577-585, 1984), and blood coagulating factor (Furie et al., Cell 53, 505-518,1988), and is thought to play important roles in extracellular coagulation and adhesion.
The vertebrate homologues of the cloned Drosophila Delta were found in chicken (C-Delta-1) and Xenopus laevis (X-Delta-1), and it has been reported that X-Delta-1 had acted through Xotch in the generation of the protoneuron (Henrique et al., Nature 375, 787-790, 1995 and Chitnis et al., ibid. 375, 761-766, 1995).
A vertebrate homologue of Drosophila Serrate was found in rat as rat Jagged (Jagged)(Lindsell et al., Cell 80, 909-917, 1995). According to Lindsell et al., mRNA of the rat Jagged is detected in the spinal cord of fetal rats. As a result of cocultivation of a myoblast cell line that is forced to over express rat Notch with a rat Jagged expression cell line, suppression of differentiation of the myoblast cell line is found. However, the rat Jagged has no action against the myoblast cell line without forced expression of the rat Notch.
Considering the above reports, Notch and ligand thereof may be involved in differentiation regulation of the nerve cells; however, except for some myoblast cells, their actions against cells including blood cells, especially primary cells, are unknown.
As mentioned above, concerning undifferentiated cells, proliferation while maintaining their specificities has not been performed. Major reasons are that factors suppressing differentiation of the undifferentiated cells have not been sufficiently identified.